DNA damage and DNA repair

Transcription

1 DNA damage and DNA repair Susan P. Lees-Miller, PhD, Professor, Departments of Biochemistry & Molecular Biology and Oncology, Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada Commonly occurring types of DNA damage: Spontaneous loss of bases Alkylation of bases Oxidation of bases UV-light induced damage: Cyclobutane dimers 6,4,-photoproducts DNA strand breaks: Natural cellular processes, exposure to radiation (cosmic, medical e.g. X-rays, radiation therapy) and some forms of chemotherapy 1

2 Estimated rates of DNA damage per human cell per day: Single strand breaks 50,000 Depurination 10,000 Deamination 600 Oxidative base damage 2000 Alkylated bases 5000 Intrastrand cross links 10 DNA double-strand break 10 Total DNA damaging events per cell per day: 60,000 Total DNA damaging events per cell per hour: 2,500 Estimate cells in human body ~ 3 x DNA damaging events per hour! Mutation is rare because of repair Over 200 human genes known to be involved in DNA repair Major mammalian DNA repair pathways: 1. Base excision repair (BER) 2. DNA Mismatch repair (MMR) 3. Nucleotide excision repair (NER) 4. DNA strand break repair pathways: Single strand break repair (SSBR) Double-strand break repair pathways (DSBR) Homologous Recombination (HR) Nonhomologous end joining (NHEJ) 2

3 Common themes in all DNA repair pathways: Detection of the lesion: protein or proteins that specifically detect and bind the particular DNA lesion Removal of the damaged DNA: glycosylases, nucleases, etc Resynthesis/Repair: Regulatory proteins: DNA polymerases, DNA ligases protein kinases etc Effects on other cellular processes: temporary halt in transcription, replication and/or cell division to allow more time for repair to take place Consequences: accurate repair: survival inability to repair: cell death misrepair: genomic instability Base Excision Repair: BER Repairs DNA bases damaged by Alkylation Deamination Oxidation Lost bases (abasic sites) Example: spontaneous deamination of Cytosine to Uracil 3

4 Base Excision Repair (BER): a simple model: Spontaneous Deamination Uracil DNA glycosylase AP-Endonuclease Spontaneous base (depurination) loss DNA Polymerase (fill) and DNA ligase (ligate) Maizels Ann. Rev Genet, 2005 BER in more detail: Involves multiple proteins Different variations of the basic pathway depending on precise type of DNA damage Different glycosylases detect different types of base damage How do DNA glycosylases detect one damaged base in a 3 billion base pair human genome? Ref: Sancar et al, 2004, Ann Rev Biochem 4

5 DNA Mismatch Repair (MMR): Corrects errors introduced during DNA replication (base mismatches, insertions/deletions) Also required for the removal of bases damaged by: Methylating agents (MNU, MNNG) Antimetabolites (6-thioguanine) and possibly Intrastrand crosslinking agents (cisplatin and MMC) Ref: Jiricny, The multifaceted mismatch-repair system, Nat. Rev. Molec. Cell. Biol., 2006, 7, DNA Mismatch Repair (MMR): Errors introduced by DNA replication Mispaired bases (base pairing errors) small insertions or deletions (slippage of polymerase) 5

6 DNA Mismatch Repair (MMR): Corrects errors introduced during DNA replication Mispaired bases small insertions or deletions Mispaired bases are detected by the MSH2/MSH6 heterodimer (MutS-a) Insertions or deletions are detected either by MSH2/MSH6 (MutS-a) OR by MSH2/MSH3 (MutS-b). Binding of MLH1-PMS1/PMS2 (Mut L) stabilizes binding of MutS a and b to the DNA mismatch/insertion deletion 6

7 MMR in more detail: Mismatch = red triangle MutSα or MutSβ binds the mismatch and recruits MuLα ATP-dependent conformational change releases the MutS/L complex from the mismatch. The complex diffuses either upstream (a) or downstream (b) of the mismatch where exonuclease I, RFC, PCNA and RPA are involved in removal of the lesion DNA polymerase delta fills the gap and DNA ligase 1 seals the ends How the system knows to repair damage on the newly replicated strand in human cells is still unknown Ref: Jiricny, Nat Rev Mol Cell Biol, 2006 DNA Mismatch Repair and Colon Cancer: Hereditary nonpolyposis colon cancer (HNPCC) the most common form of hereditary colorectal cancer. accounts for 2-7% of all colorectal cancers characterized by early onset (40-50 years), spontaneous colon cancer and increased cancer risk for endometrium, ovarian, stomach, and small intestine. >90% HNPCC patients have mutations in MLH1 (40%) or MSH2 (40%) Mutations in other MMS genes (e.g. PMS2, MSH6) are rare (1-5% of patients) Cells with defects in MMR have 1000 X greater mutation rate than MMR proficient cells and are also characterized by microsatellite instability (MSI or MIN). MIN is due to the inability of MMR defective cells to correct errors caused by DNA polymerase slippage at repetitive sequences in the genome. 7

8 Nucleotide Excision Repair: NER Repairs damage introduced by UV light Cyclobutane dimers 6,4-photoproducts Detection of UV-damaged DNA V Global NER Transcription coupled NER Repairs damage that occurs throughout genome Preferentially repairs damage in transcriptionally active genes HR23B XPC RNApol II V V Both branches converge into a common pathway involving over 20 different genes including XPA, XPB, XPD, XPF and XPG 8

9 Basic Steps in NER: Detection of lesion: Stalled RNA pol II for non- transcriptionally active genes (TC-NER) or XPC-HR23B for transcriptionally active genes (Global -NER) Common steps: Assembly of protein complex at site of DNA damage Opening of DNA strands (DNA bubble): DNA helicases Removal of DNA damage: Cut DNA strand about bases either site of lesion (endonucleases) Release of 25-32bp fragment ssdna containing the lesion Resynthesize: new DNA strand using undamaged strand as template (DNA polymerases) Seal phosphodiester backbone (DNA ligases) Global NER: 1. Damage recognition: XPC-hHR23B binds the lesion 2. DNA opening: TFIIH (XPB and XPD: DNA helicases; p62, p52, p44, p34 and others) XPA RPA 3. Incision and excision: XPG and XPF-ERCC1 (structure specific endonucleases): XPF-ERCC1 cleaves 5 to lesion XPG cleaves 3 to lesion bp piece of DNA containing the lesion is excised 4. Repair synthesis and DNA ligation: DNA polymerases delta (d) and epsilon (e), RFC, PCNA, RPA and DNA ligase 1 Ref: Park and Choi, FEBS Lett, 273, (2006). 9

10 Transcription coupled NER: 1. Damage recognition: Stalled RNA pol II Cockayne Syndrome A and B 2. DNA opening: TFIIH (XPB and XPD: DNA helicases; p62, p52, p44, p34 and others) XPA RPA 3. Incision and excision: XPG and XPF-ERCC1 (structure specific endonucleases): XPF-ERCC1 cleaves 5 to lesion XPG cleaves 3 to lesion bp piece of DNA containing lesion is excised 4. Repair synthesis and DNA ligation: DNA polymerases delta and epsilon, RFC, PCNA, RPA and DNA ligase 1 CS-A RNA pol II CS-B Nucleotide Excision Repair and Cancer Xeroderma Pigmentosum (XP): Rare genetic syndrome caused by mutation in XPA and other XP genes Characterized by UV-induced skin cancer on skin exposed to sunlight Sunlight: 90% UVA 10% UVB trace UVC Other diseases associated with defects in NER: Cockayne s syndrome and Trichothiodystrophy 10

11 DNA strand breaks repair pathways Causes of DNA strand breaks: Reactive oxygen species (ROS):generated by normal metabolic/cellular processes or external agents Errors during normal cellular processes: DNA replication, mitosis, meiosis Induced as part of naturally occurring processes: V(D)J recombination, class switch recombination Exposure to radiation: cosmic radiation, radiation during medical procedures (X-rays, CT scans, radiation therapy) Chemotherapy: many chemotherapeutics (etoposide, doxorubicin, camptothecin derivatives etc) act as topoisomerase poisons, which induce DNA strand breaks Exposure to radiation: from Lobrich and Jeggo, Nat. Rev. Cancer 2007 Most lab experiments Background dose (sea level): Transatlantic flight: Chest X-ray: CT scan: Radiation therapy: Lethal single body dose 5 µsv (higher at higher elevations) ~80 µsv ~800 µsv 30 µsv 1-2 Sv per day for days (~ 50 Sv cumulative dose) 5 Gy (Sv) 11

12 IR induces complex DNA lesions: IR-induced damage caused by direct interaction of energy with DNA (direct effects) as well as by ionization of water in vicinity of DNA (indirect effects) IR induces damage to bases, sugars and DNA backbone Produces complex DNA lesions that are lethal to the cell if not repaired Examples of types of damage: Oxidative damage (bases, sugars) Single strand breaks (SSBs): frequently with non-ligatable end groups (3 P and 3 -Phosphoglycolate) Double strand breaks (DSBs): occur when two SSBs occur on opposite strands Repair of IR induced DNA damage Base damage: BER Single strand breaks (SSBs): break in phosphodiester bond of one DNA strand SSBR pathway: SSBs detected by Poly-ADP ribose (PARP) Repaired by SSB Repair pathway: XRCC1, DNA ligase III, DNA pol beta and various end damage processing enzymes (EDP in figure) such as APE, PNK and aprataxin. Sharma and Dianov, Mol Asp Med, 28, 2007,

13 Repair of IR induced DNA damage: Double strand breaks (DSBs) Occur when have 2 SSBs bp apart on opposite DNA strands Survival misrepair Repair Cell Cycle Arrest inability to repair Genome Instability Cell death Two major pathways for the repair of DSBs in human cells Nonhomologous end joining (NHEJ): DNA-PKcs, Ku70/80, XRCC4, DNA ligase IV, XLF Artemis, PNK, DNA polymerases mu and lambda 53BP1? Tdp1? WRN? Others? Major pathway in human cells for repair of IR-induced DSBs Active throughout the cell cycle, predominant pathway in G0, G1 Does not require DNA template Potential to be error prone Required for V(D)J recombination and class switch recombination Homologous recombination repair (HRR): Mre11-Rad50-Nbs1 (Xrs2 in yeast), RPA, Rad51, Rad52, XRCC2, XRCC3, BRCA1, BRCA2 and others Predominant pathway in yeast Active in late S and G2 Requires undamaged DNA template Accurate, template directed repair 13

14 Main players in NHEJ Ku70/80: DNA-PKcs: Artemis: XRCC4: DNA ligase IV: heterodimer of 70 and 80 kds subunits, binds DSB catalytic subunit of DNA-dependent protein kinase member of PIKK family of S/T protein kinases interacts with Ku to form DNA-PK protein kinase activity required for NHEJ nuclease: interacts with DNA-PKcs scaffolding protein, interacts with DNA ligase IV stabilizes and stimulates activity of DNA ligase IV ligates DNA ends XLF: XRCC4-Like-Factor, interacts with XRCC4, stimulates activity of DNA ligase IV DNA polymerases: mu and lambda, gap filling Polynucleotide kinase (PNK): DNA phosphatase/kinase interacts with XRCC4 Working model for Nonhomologous End Joining Detection of DSB by Ku Recruitment of DNA-PKcs to form DNA-PK DNA pol mu/lambda PNK Artemis XLF Synapsis XRCC4 DNA ligase IV DNA-PK activity is required for NHEJ P 14

15 Cells that lack any of the NHEJ components are radiation sensitive + DNA-PKcs - DNA-PKcs Inhibitors of DNA-PK kinase activity radiosensitize cells Being developed as potential radiation sensitizers for radiation therapy Defects in NHEJ factors are also associated with defects in V(D)J recombination and Class Switch Recombination: Sequence specific gene rearrangement processes that occur in B (and T) cells and are required for production of immunoglobulin genes, T Cell receptor genes and functional T and B cells Inability to undergo V(D)J recombination results in lack of mature T and B cells Animals lacking NHEJ factors suffer from Severe Combined Immune Deficiency (SCID) Chaudhuri J, Alt FW. Nat Rev Immunol. 2004, 4(7):541-52). 15

16 NHEJ and DSB repair proteins are required for V(D)J and CSR: Sequence specific gene rearrangement processes that occur in B (and T) cells and are required for production of immunoglobulin genes, T Cell receptor genes and functional T and B cells Chaudhuri and Alt, Nat Rev Immunol Defects in VDJ and CSR may be linked to chromosomal translocations in B cell malignancies: Many B cell malignancies are characterized by translocation of Immunoglobulin gene promoter and proto-oncogene, leading to suggestions that defects in V(D)J and CSR may promote chromosomal translocations that are the defining characteristics of many human hematological malignancies R Kuppers, Mechanisms of B cell lymphoma pathogenesis, Nature Reviews Cancer, 5, 2005, 16

17 Homologous recombination Analysis of DNA intermediates Requires intact sister chromatid (red) End resection to produce 3 overhangs Strand invasion by 3 end DNA synthesis (red dotted line) DSBR: Second end capture Double Holliday junction Resolution of double Holliday junction Cross over or non-cross over possible OR Single strand annealing Strand displacement Annealing no cross over no Holliday junction Sung and Klein, Nat Rev Mol Cell Biol 2006 Filippo et al, Ann. Rev Biochem Protein factors involved in HR: from biochemistry and genetics Mre11, Rad50, Nbs1/Xrs2 (MRN complex): end binding, end resection Rad51: protein-dna filaments RPA: regulates access of Rad51 to DNA Rad52: interacts with Rad51 and RPA BRCA2: helps load Rad51 on DNA BRCA1: interacts with BRCA2 Sung and Klein, Nat. Rev. Mol. Cell. Biol,

18 IR-induced cell signalling and cell cycle arrest pathways Phosphatidyl inositol 3 kinase like protein kinases (PIKKs): DNA-PKcs: catalytic subunit of the DNA dependent protein kinase ATM: Ataxia-Telangiectasia Mutated ATR: ATM-, Rad-3, related DSBs DNA-PKcs ATM ATR Repair Cell Cycle Arrest Ataxia-Telangiectasia Mutated (ATM): Ataxia-telangiectasia (A-T): Autosomal recessive; compound heterozygotes Incidence 1 in ~ 40,000 to 1 in 100,000 Characterized by neurodegeneration, progressive loss of neuromuscular control, ataxia, telangiectasia, immune deficiencies, cancer predisposition (lymphoma), radiation sensitivity Over 400 mutations identified to date Mutations occur throughout the gene and are usually truncation or splicing; about 10% are mis-sense Blood relatives of A-T patients have increased risk of developing breast cancer ATM-deficient cell lines are characterized by: Radiosensitivity, radiation resistant DNA synthesis, loss of check point control, chromosomal breakage, and genomic instability ATM is also mutated in some tumour types (Mantle Cell Lymphoma) 18

19 Activation of cell signalling pathways in response to DSBs: ATM is activated in response to IR (exact mechanism of activation is still hotly debated) In response to IR (and other DNA damaging agents), ATM phosphorylates many protein targets in the cell resulting in activation of cell cycle checkpoints that cause transient arrest of the cell cycle at G1 to S, during S or at G2 to M Activation of cell cycle checkpoints may allow cells more time to detect and repair the DNA damage ATM phosphorylates the tumour suppressor protein p53, which regulates cell cycle arrest at G1/S and cell death by apoptosis 19

20 DNA damage induced activation of p53 Chene, Nature Reviews Cancer, 2, 102 (2003) ATM substrates: BRCA1 and BRCA2 BRCA1 and BRCA2 genes: discovered in 1990s as Breast and ovarian cancer susceptibility genes Mutations in BRCA1 and BRCA2 account for about 60% of hereditary breast cancer. However, only 5 to 10 % of all breast cancers are hereditary: most are sporadic The causes of sporadic breast cancer are not well understood. Annual rates of breast cancer (US): 215,000 women; 1500 men Gene and protein sequences had no distinguishing features that suggested what BRCA1 and BRCA2 actually do! 20

21 BRCA1 and BRCA2 are involved in the DNA damage response: BRCA2: interacts directly with Rad51 required for HR BRCA1: interacts with BRCA2, p53, Rad51: involved in HR and possibly NHEJ phosphorylated by ATM and Chk2 in response to DNA damage. Phosphorylation required for cell cycle checkpoints BRCA1 and BRCA2: Recruited to sites of DNA damage after IR (IRIF) Rev: Yoshida and Miki: Cancer Sci 2005 Summary DNA damage happens: caused by endogenous and exogenous sources Cells have multiple and complex pathways to detect and repair each specific type of DNA damage Major repair pathways in human cells (BER, MMR, NER and the strand break repair pathways: SSBR, HR and NHEJ) Perfect repair would result in genome stability Imperfect repair can promote genetic divergence but also cause genomic instability From Friedberg (2001) Nat. Rev. Cancer, 1,

22 Understanding DNA repair pathways has lead to greater understanding of some human diseases DNA repair protein MSH2, MLH1 XP proteins ATM Nbs1 Chk2 Artemis DNA-PKcs DNA ligase IV FA proteins Apratxin DNA repair Pathway MMR NER DSB signalling DSB signalling, HR DSB signalling NHEJ NHEJ NHEJ DNA crosslink repair, HR? SSB repair Disease Syndrome HNPCC XP skin cancer A-T; predisposition to breast cancer Nijmegen Breakage Syndrome Breast cancer RS-SCID RS-SCID in mice dogs and horses Lig4 syndrome Fanconi Anemia AOA1, microcephaly Adapted from O'Driscoll M, Jeggo PA..Nat Rev Genet Understanding DNA repair pathways could lead to a better understanding of the causes of genomic instability: Chromosome translocations are a hallmark of genomic instability Cancer cells have highly unstable genomes characterized by chromosome duplication, chromosome loss, and chromosome translocations. Spectral Karyotype analysis: Karyotype of a normal cell Karyotype of a cancer cell How do chromosomal translocations occur? Are these caused by aberrant DNA repair mechanisms? 22

23 Better Response to Radiation therapy? Approximately half of all cancer patients are treated with radiation therapy Some patients respond top treatment and survive Others get same treatment but suffer from poor treatment outcomes Understanding DNA repair pathways could also lead to ways to predict radiation response in cancer patients Immunohistochemistry of protein levels of proteins involved in DNA repair, cell survival as well as hypoxia and angiogenesis etc could help predict whether tumours will respond to radiation (or other DNA damaging agents) or not Similar for mrna expression levels, microrna profiles, SNPs Reduced side effects of radiation therapy? Understanding DNA repair pathways could also lead to development of novel radiosensitizers Specific inhibitors of DNA-PK and ATM kinase activity sensitize human cell lines to IR and chemotherapeutics Confirmed in animal models (Zhao et al, Cancer Research 2006) Could they be of use as radiosensitizers in cancer patients? 23

24 Understanding DNA repair pathways could lead to novel cancer therapies Some cancers are characterized by defects in DNA repair proteins Examples: Mutation of BRCA1 and BRCA2 in hereditary breast cancers Mutation of loss of ATM in mantle cell lymphoma, B-CLL, and possibly some lung and gastric cancers Hypothesis: One DNA repair pathway is compromised; so cancer cells rely more on other repair pathways Prediction: If inhibit the alternative pathway will this kill the tumour cells? Yes! BRCA1/BRCA2 defective breast cancer cells (defective DSB repair; HR and ATM dependent pathways) are highly sensitive to inhibition of PARP (SSB repair) Farmer et al, Nature 2005; Bryant et al, Nature, 2005 The Lees-Miller lab: Effects of radiation and other DNA damaging agents on cells Mechanism of NHEJ Role for DNA-PK and ATM in the DNA damage response Mechanisms of tumour radiation resistance and radio sensitivity: biomarkers of radiation response Developing novel therapies based on understanding the molecular details DNA repair pathways Web site: 24